The stereospecific reduction of carbonyl groups can be used to produce chiral alcohols. Enantiomerically homogeneous chiral secondary alcohols are useful intermediates for pharmaceuticals, and may be prepared from ketones using NADH/NADPH-dependent secondary alcohol dehydrogenases. Several biochemical and chemical approaches have been employed in the synthesis of enantiomerically pure alcohols. These approaches include stereospecific chemical reduction of ketones, enzymatic hydrolysis of racemic esters, and enzymatic esterification of racemic alcohols. Notably, microbial enzymes have been used for the synthesis of chiral alcohols at laboratory, pilot, and production scale (C. J. Sih and C.-S. Chen, 1984, Angew Chem. Int. Ed. Engl. 23:570-578; O. P. Ward and C. S. Young, 1990, Enzyme Microb. Technol. 12:482-493). These synthesis reactions are typically carried out using resting cells, isolated enzymes, and/or cloned and overexpressed enzymes.
In particular, Rhodococcus erythropolis NADH-dependent carbonyl reductase has been used with a wide range of substrates, including 2-ketones, 3-ketones, keto-esters, and aromatic ketones (T. Zelinski and M.-R. Kula, 1994, Bioorg. Med. Chem. 2:421-428; T. Zelinski et al., 1994, J. Biotechnol. 33(3):283-92). Additionally, Baker's yeast has been widely used for the asymmetric reductive biotransformation of a variety of 2-ketones and 3-ketones (R. Csuz and B. I. Glanzer, 1991, Chem. Rev. 91:49-97; R. Devaux-Basseguy et al., 1997, Enzyme Microb. Technol. 20:248-258; W. Hummel and M.-R. Kula, 1989, Eur. J. Biochem. 184:1-13; W. Kruse et al., 1996, Recl. Trav. Chim. Pays-Bas 115:239-243; T. Loviny et al., 1985, Biochem. J. 230:579-85; W. F. H. Sybesma et al., 1998, Biocatal. Biotransform. 16:95-134; O. P. Ward and C. S. Young, 1990, Enzyme Microb. Technol. 12:482-493).
Similarly, Gluconobacter oxydans cells have been used in the reduction of various ketones to (S)-alcohols with high enantiomeric excess (P. Adlercreutz, 1991, Enzyme Microb. Technol. 13:9-14; P. Adlercreutz, 1991, Biotechnol. Lett. 13:229-234). In addition, G. oxydans 2-ketoreductase has been purified, and the purified polypeptide has been partly sequenced (V. Nanduri et al., 2000, J. Indust. Microbiol. Biotechnol. 25:171-175). However, large-scale synthesis of S-(+)-2-pentanol requires a large cell mass, i.e., a ratio of 2-pentanone to cell mass of 1 kg:50 kg. In accordance with the present invention, G. oxydans 2-ketoreductase was purified and cloned for overexpression in Escherichia coli. The disclosed 2-ketoreductase expression system thereby allows industrial production of S-(+)-2-pentanol and other chiral alcohols.